Hi folks,
We are aiming to kick off a genome sequencing project on an anole (lizard) species, Anolis distichus (genome size somewhere between 1.8 and 2.5Gbp). We've been researching "best practices", and the general consensus I am coming across is a combined coverage (of shorter and long inserts) of about ~100X, having long insert mate-pairs (~10-20kb+), and using SOAP or ALLPATHS as an assembler seems to lead to a decent assembly. However, recently, we've come across DISCOVAR de novo and Platanus as potential assembler options as well.
Obviously, choice of assembler is going to affect our library prep, so I wanted to canvass the community to see if anyone had any updated thoughts on current best genome sequencing practices (most of the posts/genomes I found were initiated in 2014 or before).
Resources:
-- A 'relatively' inbred individual for sequencing, and high quality DNA extracted from it
-- One of its congeners, A. carolinensis has been sequenced
-- We'll have a transcriptome for the individual we sequence
-- $5-7k for whole genome sequencing costs
Our current feeling is aiming for 2*250bp reads of ~450bp insert sizes (Illumina) will allow us to use DISCOVAR denovo, and if along with the transcriptome, that doesn't give us a "pretty enough" assembly (we are looking for a very high quality draft as we are interested in specific chromosomal regions involved in divergence across the genus), we could then add in mate-pair (and potentially pacbio?) if we needed to, and try ALLPATHS.
Do people have strong thoughts on how they would attack the project with the same resources? We would love to hear from you if so. Full disclosure: also asking this question on seqanswers, so if I get any answers there that I think people here would like to hear I'll make sure to share.
Cheers!
I think 40~60-fold of 2*250bp reads of ~450 bp insert size should be suitable at the first stage. It is the recommended coverage for both ALLPATHS and DISCOVAR. Mate-pairs or pacbio data can significantly benefit assembling, however, they are not supported by DISCOVAR to date (but maybe supported in the near future). So I recommend to generate mate-pairs or pacbio data if the cost can be covered. Then, if the resulted assembly at the first stage isn't very good, those data can be used by ALLPATHS to enhance the assembling. Once they are acceptable for DISCOVAR, you can also have a try.
For a genome that size, you should really try to include some long reads, either PacBio or Illumina Synthetic Long Reads. Actually, I would rather start with only the Illumina Synthetic Long Reads, and from there sequence either very long insert mate pairs and/or get some PacBio reads.
Thanks for the thoughts Diego - just to make sure I am understanding your perspective: you would do PacBio/Illumina Synthetic Long Reads in addition to, not instead of, short-insert Illumina libraries?
Alana, I would start with the long reads (Illumina). This is what I am doing for Sugarcane (complex polyploid and aneuploid genome), and it is working very well. Others have used a combination of long reads from Illumina and PacBio with very good results as well.
Thanks for your feedback folks - just in case anyone is interested, the recommendations I got from seqanswers (http://seqanswers.com/forums/showthread.php?p=171783#post171783) were:
-- People liked the no amp PE libraries. The Illumina PCR-free kit works well.
-- Depending on assembler used, 2*250bp won't give you much improvement over 2*100bp
-- DiscovarDeNovo has pretty hefty memory requirements (between 800G and a TB of RAM for an assembly with ~250M 250bp reads )
-- ABySS-PE seems really good at animal and fungal genome assemblies. It can handle 250 base reads fine. (ALLPATHS probably could also.)
-- Mate-pair libraries really help. Even the cheap-to-make no-gel TruSeq Nextera Mate Pair libraries.
We have used 2x300 bp amp-free MiSeqs (much more expensive) and it works well. So 2x250 on the cheaper HiSeq is a good idea.
Then 2Kb and 10Kb mates and Allpaths-LG.
Make sure all the DNA is from one individual. But $7K won't be enough.
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